chapter 9 (mod 3)
Recombinant DNA techniques
-bacteria are the workhorses of modern biotechnology -bacterial plasmids
Discuss agricultural applications of rDNA technology and the concerns of the use of genetically modified organisms to human safety, its impact on the environment and the ethics of using altered animals.
- today, DNA technology is quickly replacing traditional plant-breeding programs -scientists have produced many types of GMOs, organisms that have acquired one or more genes by artificial means -many staple crops in the U.S. such as corn, cotton, and soybean are genetically modified. -corn has been genetically modified to resist insect infection -genetically modified is a very general term, but public tends to only be concerned when it pertains to food crops -examples of GMO crops are 'golden rice' which is genetically modified to have beta-carotene to give vitamin A and strawberries genetically modified to have natural antifreeze so they can survive cold weather -animals are also genetically modified to have human genes ie for human hemoglobin to be isolated and used in blood transfusion -a crop example is Bt corn and cotton which carry toxin-producing gene from B. thuringiensis. this toxin kills insects that eat plants Also found a Ti plasmid in Agrobacterium tumefaciens that integrates into the plant genome and causes a tumorlike growth on plants. Now are often used to introduce rDNA into a plant for experiments involving plants. Concerns: -concern with accidnetal release of altered organisms, impact of modified organisms on non-target organisms (superweeds). -potential for creating genetically modified biological weapons or new pathogens -controversy over GMO crops, concern if safe for us to eat and the environment, we don't know long term effects. -there is now a movement for labeling and identifying GMO containing foods -there are concerns for altering animals for human use and privacy of individual's genetic information. Many peoples DNA are already online from ancestry tracking websites.
Outline the steps of PCR and note the examples of its uses as discussed in class.
-aka Polymerase chain reaction -it is a technique to quickly and precisely copy any segment of DNA -this is used to amplify NDA to detectable levels for analysis, clone DNA for recombination, sequence DNA, diagnose genetic disease, and detect pathogens. -PCR requires target DNA, specific primers, taq DNA polymerase, nucleotides, and a machine called a thermocycler (cycles through diff temperature changes) -primers (short, chemically synthesized, single-stranded DNA) are complementary to sequences flanking the target sequence, it is important they are specific to be complementary to the exact segment of DNA you are targeting -Heat stable Taq DNA polymerase copies the target DNA sequence -it is a three-step process that doubles the amount of DNA at the end of each cycle One PCR cycle: -double stranded DNA put in high heat which denatures it into single strands, 94-95 degrees C -cycled to a second temperature at which the primers will bind and anneal to the DNA, 50-56 degrees C -cycled to third temperature for extending stage; taq (thermus aquaticus, from organism that is a hyperthermophile so it can work at high temps) DNA polymerase will start at the primers and add complementary bases to the template DNA strands, 72 degrees C -programmed to go through 30-40 cycles usually and makes lots of DNA
Describe how colony hybridization can be used to identify clones carrying a desired gene.
-colony hybridization uses a DNA probe to identify cells carrying cloned genes. -specific fluorescence labeled probes are complimentary to the gene of interest and bind to them. -you take colonies of recombinant bacterial cells, take a filter paper and press it onto the place to make a replica of the plate. -the filter is then treated with detergent to lyse the bacteria. -the filter is then treated with sodium hydroxide to separate DNA into single strands -fluorescent probes are applied, they are specific ie to human insulin gene and will only anneal to colonies which have the specific gene. -if it does anneal, it will bind to the DNA and light up the colony so you can identify which colonies carry the gene of interest. -you know the location of the colony on master plate so you can then utilize the bacteria with needed gene/locate them.
Blue-White screening
-one method of selecting recombinant bacteria -all the recombinant bacteria with ampicillin-resistance gene on plasmid (some with recombinant insert, some without) are plated onto a NA plate containing ampicillin. -The bacteria with the recombinant plasmid will not produce the enzyme B-galactosidase which allows them to hydrolyze B-galactosidase substrate. Normal bacteria make this and when they break down substrate they produce an indigo compound that makes the colonies blue -Recombinant DNA bacteria will not produce the enzyme, therefore not turning blue and will make white colonies that are distinct from the blue ones and can thus be identified.
Discuss how DNA containing a gene of interest may be obtained from a genomic library, cDNA and DNA synthesis machine.
1. Gene libraries are made of pieces of an entire genome stored in plasmids or phages. Made by the genetic cloning technique -a genomic library is a collection of clones containing different DNA fragments OR a collection of clones representing all genes from a single chromosome library has cells with different fragments to all represent the DNA, you can go find specific fragment you want/have them all on hand in collection 2. cDNA is made from mrna by reverse transcriptase -called complementary DNA -cDNA is made from eukaryotic mRNA using reverse transcriptase. the reverse transcriptase is an enzyme which will convert RNA into DNA, but it is copied in reverse so it is called complementary DNA -for eukaryotic genes, mRNA has introns removed so the cDNA generated can be cloned and expressed in bacterial system -bacteria lack enzymes to process eukaryotic pre-RNA which it is important to process eukaryotic DNA like this to bypass the issue of RNA processing 3. synthetic DNA is made by a DNA synthesis machine In vitro (in a test tube) synthesis of short sequences is possible, about 200 nucleotides. However, the sequence must be known.
Describe five ways of getting DNA into a cell.
1. Transformation- cells take up DNA from surrounding environment. Done with test tube, species of bacterium that naturally transforms, and DNA. However, only a small number of cells (bacterial cells) do this so for other organisms you have to make them be able to take in DNA through these other techniques: 2. Electroporation- application of electrical current forms pores in cell membrane to allow DNA to be taken up. Makes cells competent so DNA can be taken up to make recombinant cells 3. Protoplast fusion- cell wall is removed from bacteria to allow cells to fuse together and recombine DNA 4. Microinjection, usually for eukaryotic animal cells. tiny needle injection 5. Gene gun, bombard little DNA coated beads into cell, usually for plant cells.
Discuss the use of rDNA technology in therapeutic and scientific applications.
Applications of recombinant DNA technology are in medicine, basic science, forensics, and agriculture. -wide-ranging application of this technology examples of very important medical products from rDNA technology: -human insulin -human growth hormone human gene therapy: -a recombinant DNA procedure that seeks to treat disease by replacing defective or missing genes with normal functioning genes (SCID, CF (cystic fibrosis), hemophilia) in afflicted patient. -SCID is a fatal inherited disease caused by a single defective gene that prevents the development of the immune system. SCID patients quickly die unless treated with a bone marrow transplant or gene therapy. Since 2000, gene therapy has cured 22 children with inborn SCID but unfortunately caused 4 of the patients to develop leukemia. Recently, new gene therapy has cured 10 newborns with SCID. Genome Mapping and sequencing (genomics): compares total genomes of different organisms, map genomes of pathogens to find potential vaccine targets. -human genome project recently completed and mapped and sequenced the entire human genome. from this they discovered less than 2% of genes actually code for proteins, got info about embryonic development, evolution, and disease-associated genes with will aid diagnosis and treatment -human proteome project is ongoing and aims to mapp all the proteins expressed by human cells DNA fingerprinting: -used in forensics and paternity testing -each person is genetically unique unless they are an identical twin -due to this, your nucleic acid sequence is unique, cutting each persons DNA with same restriction enzymes will produce different sized fragments -gel electrophoresis is used to observe banding patterns that make up each persons unique DNA fingerprint. -useful for murder evidence -useful for tracking pathogens as well(ie track infectious disease and the source of outbreak) Microarrays: -have DNA sequences on slide, or microarray - you put fluorescently labeled DNA into specimins, and if complementary will bind and show if gene is present. used in medical diagnostics.
Compare and contrast biotechnology and recombinant DNA (rDNA) technology.
Biotechnology- the use of microorganisms, cells, or cell components to make a desired product. This is not a new thing, traditionally has been used for industrial and culinary applications: to brew beer and wine, collect antibiotics (penicillin), vitamins, and enzymes made by microbes Recombinant DNA (rDNA) technology- a branch of biotechnology that makes use of modern tools and techniques to artificially modify genes to produce desired proteins. The difference is recombinant DNA technology is the modern version, modern tools and techniques to artificially modify genes to produce desired proteins. Whereas in biotechnology, the microbes used already make the certain enzymes for the end product you want so you are just making use of them as they are.
Discuss the use of E. coli, B. subtilis, S. cerevisiae, plants and mammalian cells in expressing gene products.
Cloning in Escherichia coli: -after putting gene of interest into vector, made more in PCR, screened for it, you must grow it up in large quantities -E. coli is very commonly used as a bacterial organism for gene expression -this is because E. coli is easily grown and its genomics are known -however, some disadvantages are you must eliminate endotoxin products from their cell wall/the product made because even in small quantities it can cause endotoxic shock or fever. Additionally, E. coli does not naturally secrete protein products so cells must be lysed to harvest the product. Bacillus subtillus: -are Gram-positive and secrete gene products, they lack endotoxin, grow well, and genomics of it are known. Saccharomyces cerevisiae: -a yeast, since it is eukaryotic it expresses foreign eukaryotic genes well which is an advantage -it is easily grown and its genomics are known -it continuously secretes gene product Plant cells and whole plants: -express eukaryotic genes easily -plants are easily grown, large-scale, and low cost Mammalian tissue culture cells: -may express eukaryotic genes easily -can make products for medical use -harder to grow, requires more training and technology to grow but often still method of choice for products of pharmaceutical or medical use.
Summarize the general overview for the cloning of genes in bacteria.
Gene cloning is the production of multiple copies of a gene. bacterial plasmids are used for gene cloning. -plasmids are small, self-replicating, circular DNA molecules; non-essential for growth; often carry genes for virulence or antibiotic resistance; they can also act as vectors (DNA carriers) that move genes from one cell to another. E. coli with plasmids, plasmids can be isolated, digested with a restriction enzyme (cut open), DNA ligase and a particular human gene ie insulin gene can be added and the DNA ligase will join the gene with the plasmid, making a recombinant plasmid. The recombinant plasmid is then mixed with a bacteria and the plasmids enter bacterial cells through the process called "transformation" the E. coli then grows containing the recombinant plasmids and the DNA is transcribed and expressed
humulin
The world's first genetically engineered pharmaceutical product sold, in 1982. It is human insulin produced by genetically modified bacteria it is used today by more than 4 million people with diabetes. Previously insulin was extracted from animal tissues but humulin can be produced in large quantities, cost effectively, without impurities and the protein is made from a human gene put into a bacteria. Goal of DNA recombinant technology is increase range of products and make them safer, more cost effective, and efficient. Why is it possible to make a human gene product in a bacterium? there is a universal genetic code; the bacterium understands and knows how to read the human gene and to express it by making those proteins.
staggered vs blunt ends
done by restriction enzymes, each recognizes a specific sequence. staggered cutters recognize a 4 nucleotide base sequence and cut before it. Since DNA is antiparallel, this results in cutting at different spots on each strand so there is an overhang of 4 bases on one strand, known as a "sticky end". DNA cut by sticky end enzymes will re-anneal with DNA cut by the same enzyme because they will be complementary sequences. blunt cutters find a site and cut straight across resulting in not a sticky end, but a blunt end. blunt end restriction enzymes create blunt ends that can be joined to ANY blunt end cut DNA.
Identify the roles of restriction enzymes, vectors and DNA ligase in making rDNA. Describe the properties of restriction enzymes and vectors noting examples from lecture.
restriction enzymes: -enzymes naturally made by bacteria to degrade invading foreign viral DNA -enzyme recognize and cut specific nucleotide sequences -they create staggered "sticky" ends or blunt ends which can recombine with other similarly cut ends -the bacteria's own DNA is methylated to protect from digestion by these enzymes -scientists use restriction enzymes to combine DNA fragments into a vector, by cutting up pieces of DNA to be joined together Vectors: -plasmids, have their own origin of replication -we pick ones with a recognizable marker for selection to use, such as ampicillin resistance which you can identify by what grows on media with ampicillin. -within the lacZ gene, there are different specific restriction sites so you can decide to use one of the restriction enzymes, cut the plasmid vector with that enzyme, cut the target DNA with the same enzyme, and then these two pieces of DNA will be complementary and have the potential to re-anneal with one another. -helps make recombinant plasmid DNA ligase: -